1. Field of the invention
The present invention relates to an apparatus for analyzing cells by means of flow cytometry.
2. Description of the Prior Art
In flow cytometry, a sample including cells (or particles like cells) conjugated, for example, by use of fluorescent dye or antibodies is passed together with sheath fluid into a crystal flow cell. The sample is wrapped in a pressurized sheath flow so that a thin, stable stream of the sample (laminar flow) flows through the center of the flow cell (hydro-dynamic focusing). The cells line up within stream and the flow past a focused laser beam (the sensing zone) at a constant speed. At this point, scatter lights and fluorescence from the cells are measured simultaneously by some independent sensors. The computer analyses the strength of these signals and uses it to classify the cells.
As cell analyzer by flow cytometry, there has been known an apparatus including a flow cell for producing a thin stream, a light source (for example, a laser device) to apply a light beam onto cells flowing through the flow cell, a sensor or detector for detecting light information of cell on which the light beam is irradiated so as to convert the information into electric signals, and a computer achieving operations such as an analysis process of the light information of the cells thus represented in the form of electric signals.
In this cell analyze apparatus of the prior art, a sample in which cells conjugated a fluorescent dye or antibodies are floating is supplied into the flow cell together with sheath fluid. A sheath flow is then formed in the flow cell such that owing to the hydro-dynamic focusing effect, the cells are arranged in a line along a center axis of the flow cell.
When a light beam is applied onto the cells, there are developed scatter lights and fluorescence such that the intensity of the lights and fluorescence are detected as parameters constituting the cell light information by means of light detectors such as a photoelectric multiplier.
Incidentally, there exists a case in which cell analysis is desired to be effected on one cell population selected from a plurality of cell populations included in the sample. For example, in a case of an analysis to be conducted on lymphocyte subsets or phagocytosis of human blood cells, whole blood is employed as a sample. That is, in this situation, incubated with a monoclonal antibody conjugated with a fluorescent dye [in a case of two-color analysis, for example, an OKT4 monoclonal antibody conjugated with fluorescenin isothiocyanate (FITC; green fluorescence) and an OKT8 monoclonal antibody conjugated with phycoerythrin (PE; red fluorescence)] After it is caused to react upon whole blood so as to be thereafter subjected to hemolysate a sample is thereby prepared.
The laser beam is irradiated onto each cell flowing through the flow cell so as to detect by means of detectors which measure four parameters, namely, an intensity of forward light scattering I.sub.0 (of scatter light in a direction along the optical axis of the radiated beam), an intensity of 90.degree. or right angle light scattering (of scatter light in a direction orthogonal to the optical axis of the radiated laser beam), an intensity of green fluorescence I.sub.g, and an intensity of red fluorescence I.sub.r, thereby obtaining light information of the cell (to be called cell light data in some cases). The sample includes, in addition to lymphocytes, other substances such as monocytes and granulocytes and hence it is necessary to discriminate the data related to lymphocytes from other cells.
For this purpose, there has been known a method called a window method in which the cell light information associated with a desired cell population is discriminated and is gathered. (For details, refer to the Japanese Patent Unexamined Publication (Kokai) No. 62-134559, for example. ) According to the window method, in a space including a coordinate system constituted with one or more parameters selected from the light information items of the cells, the operator establishes an analysis region or area called a window such that light information of the cells belonging to the area is collected as the light information of the objective cell population.
For example, in lymphocyte subset analysis, there are adopted two parameters including the intensity of forward light scattering I.sub.0 to represent cell size and the intensity of right angle light scattering I.sub.90 to indicate complexity of cell internal matter so as to draw a cytogram in which the abscissa and the ordinate designate the values of I.sub.90 and I.sub.0, respectively. In this diagram, the values of I.sub.90 and I.sub.0 are normalized depending on the maximum values measured so as to set a maximum value of the scale to 256 (eight bits). The values are represented in the unit of channels (ch); moreover, b, c, and d respectively designate distributions of lymphocytes, monocytes, and granulocytes, respectively. In the graph, a stands for a distribution of debris, which includes substances such as membrane components of red blood cell and is usually removed at the noise threshold.
For analysis of lymphocytes, a reference sample (a sample of a person of a normal health) is employed so as to set a window e as indicated by double-dot-and-dash lines in FIG. 1. Data related to lymphocytes associated with the window e is selected (extracted) from the data gathered through the measurement. The data thus selected for lymphocytes is subjected to computations of the intensity I.sub.g and I.sub.r of the green and red fluorescence, respectively so as to attain the positive ratios of the reaction with a monoclonal antibody conjugated with a fluorescent dye such that the results are displayed on the CRT or are printed out on a sheet of paper by means of the printer.
However, according to the window method above, it is necessary in some cases for the operator to change the window depending on a sample so as to collect the light information of the objective cell population. For example, in lymphocyte subset analysis, since the location, size, and contour or shape of the distribution b of lymphocytes shown in FIG. 1 vary depending on the sample, the operator is required to change the window e in a corresponding fashion. Such a change of window prevents an automatic measurement of a great number of samples from being conducted with high efficiency.
In order to cope with such a difficulty, the present applicant has already filed an application of an automatic cell analyze apparatus in which the measurement is automatically carried out without necessitating the operator to establish the window (Japanese Patent Application No. 62-22884: Kokai No. 63-191043). In accordance with the cell analyzer above, one or more parameters selected from the cell light information items are employed to generate histograms such that minimal points (associated with the smallest frequency value in the distribution) are detected from the histograms so as to subdivide the cell populations to establish an analysis area including one or more subdivided regions or partitions, thereby collecting light information of cells belonging to the analysis area as the light information of the objective cell population. In this description, the minimal point does not indicate a minimal point defined in a sense of mathematics, namely, indicates a portion of a valley appearing between adjacent peaks in the frequency distribution.
For example, in the case of the lymphocyte subset analysis, as can be seen from FIGS. 2a and 2b, there are produced histograms associated with the intensity of right angle light scattering I.sub.90 forward light scattering I.sub.0 in which the ordinate designates the number n of cells. There are detected minimal points p.sub.1, p.sub.2, and p.sub.3 of the histogram of I and minimal points p.sub.4 and p.sub.5 of the histogram of I.sub.0. These minimal points p.sub.1 to p.sub.5 are represented in a cytogram related to I.sub.90 and I.sub.0 so as to obtain partitions or fractions indicated with broken lines in FIG. 1. Since the distribution of lymphocytes is included in the fraction B, there are retrieved, from the light information items of all the measured cells, light information items of cells belonging to the fraction B, namely, of cells for which I.sub.90 is at least p.sub.1 and at most p.sub.2 and for which I.sub.0 is at least p.sub.4 and at most p.sub.5, thereby collecting the light information of lymphocytes.
However, depending on samples, particularly, in a case of blood of a patient, there cannot be extracted any expected minimal points from the histograms produced with respect to one or more parameters above and hence the light information of the objective cell population cannot be attained in some cases. For example, in the lymphocyte subset analysis, there exists sometimes such case that the above minimal points p.sub.a or p.sub.1 cannot be detected in a histogram of the intensity of right angle light scattering I.sub.90 depending on conditions, histograms of such case being shown in FIGS. 8a and 8b which will be described later.
On the other hand, even if such minimal points are detected and there is determined a fraction containing the objective cell population, it is required to retrieve data items of all the measured cells by use of the two parameters so as to gather the light information of the objective cell population, which leads to a problem that a considerably long period of time is necessary for the data collect processing.
On the other hand, in the cell analyze apparatus of the prior art above, cell light information collect means gathers the light information of the objective cell population. However, as shown in FIG. 1, the fraction is a region having a rectangular shape in the cytogram; in consequence, the fraction is not completely matched with the shape or contour of the distribution of the objective cell population and hence light information of unnecessary cells are included in the attained light information of the objective cell population, namely, there arises a problem that the analysis precision is lowered.
As a method to determine an analysis area more suitably matched with the contour of the distribution of the objective cell population, there has been known the contour trace method, which however requires a long period of computation time and hence is not suitable with respect to the efficiency of the cell analysis.
Generally, the sample includes, in addition to the cells, other substances such as dust and dirt in a small amount; furthermore, the sheath fluid also includes a slight amount of dust. When such dust passes through the flow cell, unnecessary information, namely, a noise appears in the cell light information. For example, in a case of lymphocyte subset analysis, as shown in FIG. 3a, substances such as membrane components of erythrocytes remained in the sample as a result of hemolysis may appear as a ghost (debris) a as described above, or the dust in the sample or sheath fluid may be detected as a noise ni.
To overcome this difficulty, in the cell analyze apparatus of the prior art, there is disposed a noise threshold circuit in a signal processing circuit to process signals supplied from the light detectors or photosensors such that the noise threshold levels Nh and Ns are established as shown in FIGS. 3a and 3b so as to remove the ghost a of red blood cell and the noise ni due to the dust, thereby guaranteeing the reliability of the cell analysis. In order to set the noise threshold values, the operator inputs threshold levels to the computer, which in turn transfers the received levels to the noise threshold cirucit. Namely, the computer operates only as an interface between the operator and the noise threshold circuit; in other words, in the conventional cell analyzer, it can be considered that the noise is removed by means of the hardware system.
In this situation, however, in a case where a great amount of samples are to be processed in a sequential fashion, for example, when an automatic sampler or an auto-sampler automatically supplying samples is used, due to the ghost of red blood cell and the dust in the sheath fluid, there is frequently required an operation to rearrange the noise threshold levels again, which leads to a problem that the efficiency of the inspection is reduced. In addition, if the change of the setting of the noise threshold levels is mistakenly ignored or if the setting change is inappropriately achieved, there arises a problem that the reliability is lowered in the cell analysis.
Recently, in the cell analyze apparatus above, in order to measure a large amount of samples efficiently, there has been considered an introduction of a so-called auto-sampler, which automatically supplies samples to the measurement system. Furthermore, with a provision of the auto-sampler, a direct contact can be avoided between the samples and the operator, which is favorable with respect to the prevention of bio-hazard.
However, there exist many factors exerting influences onto the positive ratio and hence the measurement conditions vary among the sample processing methods. For example, since the reaction between the various monoclonar antibodies and cells characterizes the results measurement, even when the identical detector is employed, the measurement cannot be conducted by use of the same detection gain. Moreover, due to the difference among the types of linkages between the monoclonal antibodies and fluorescent materials, there is required a correction to be effected when an intensity of the fluorescence is detected.
In consequence, for each sample, it is necessary to select and to set an appropriate measurement condition; however, the selection and setting operation requires knowledge gathered through a long experiece, and hence, conventionally, the operator achieves the select and set operation while monitoring the data. This job however necessitates an experienced skill and a considerable volume of labor; in consequence, the reliability of measured results cannot be increased and a great amount of samples cannot be efficiently subjected to the measurements. As a result, even if the auto-sampler is adopted so as to automatically conduct only the operation to supply the samples, the operation to select and to set the measurement condition is kept unchanged like in the case of the conventional system, and hence it is impossible to increase the efficiency of the measurements and to improve the the reliability of measured results.